The goal of this renewal R15 proposal is to gain further insight into the role of the redox-sensing transcription factor SoxR in the antibiotic-producing bacterium Streptomyces coelicolor. Members of the Streptomyces genus produce the majority of known antibiotics, but the mechanisms that allow these organisms to adapt to endogenous antibiotics are not well understood. We have evidence that SoxR may play a role in this adaptation. In S. coelicolor, SoxR-regulated genes are expressed in response to the production of the redox-active antibiotic, actinorhodin. SoxR-regulated genes encode an ABC-transporter and several putative proteins with homology to antibiotic-tailoring enzymes. A ?soxR mutant overproduces actinorhodin compared to the parental strain, suggesting that SoxR regulates mechanisms that normally reduce production of this redox-active metabolite thereby allowing this organism to avoid self-toxicity. We propose that SoxR-regulated proteins modify toxic intermediates in the actinorhodin biosynthetic pathway and then expel them from the cell. As such, we expect to find differences in the metabolites produced by wild type and ?soxR cells, which we will determine using a combination of high pressure liquid chromatography (HPLC) and mass spectroscopy (MS) analyses. In order to identify the pathway intermediates that serve as substrates for SoxR-regulated enzymes, we will create blocks in the actinorhodin biosynthetic pathway (by mutations) in the wild type and ?soxR backgrounds, to force the accumulation of specific intermediates. If any of these intermediates are metabolized by SoxR-dependent mechanisms, their levels will be higher in SoxR-deficient cells compared to SoxR-containing cells, and this wil again be detected via HPLC-MS analysis. The expression of the SoxR- regulon in the act biosynthetic mutants will be monitored by quantitative real-time PCR to determine if SoxR senses a redox-active precursor of Act, and thus induces detoxification genes to accompany or precede the production of toxic molecules. To obtain a better understanding of the mechanism via which SoxR mediates its function, individual SoxR-regulated genes (which directly execute SoxR's proposed detoxification function) will be deleted, and the mutants characterized genetically and biochemically. In addition, we will construct a strain that is deficient in both SoR and a putative actinorhodin exporter (not part of the SoxR regulon) and assess the fitness of the resulting mutant. Together, these studies will enhance our understanding of how SoxR helps antibiotic producers adapt to toxic endogenous metabolites. Finally, as an AREA proposal, the research will train students in the design and execution of hypothesis-driven experimentation and data analysis.